Stroke is the name given to sudden neurological deficits most commonly caused by obstruction or haemorrhage of an artery supplying a region of the brain. Any region of the brain can be affected.
There are an estimated twelve million survivors of stroke in the principal European, American and Japanese markets, with the number of new cases in these markets growing at 7 percent per annum. Approximately 30 percent of stroke patients require ongoing nursing care, estimated to cost between $25 billion to $30 billion per annum in the US alone.
The 20 percent of survivors with moderate or severe disability after stroke are potential candidates for neural stem cell transplantation therapy. Existing drug treatments serving these patients are limited and seek to address the effects rather than the cause of the condition.
Huntington's disease is an uncommon, inherited, progressive and fatal neurodegenerative disorder. In the US, approximately 35,000 patients show overt signs of the disease, with a further 75,000 carrying the abnormal gene. There are no existing treatments for the disease.
Dementia is the most devastating and costly age-related disorder. The most frequently encountered type is senile dementia of the Alzheimer's type (AD), although some (but not all) treatments for Alzheimer's may eventually prove applicable to other dementias. Currently, the cost of caring for Alzheimer's patients, who can no longer safely care for themselves, is estimated to approach $100 billion in the US. Age itself is a risk factor for Alzheimer's, the prevalence of Alzheimer's in the population doubles from age 65 to 75, and again from 75 to 85, at which point 35% show signs of AD. With the ageing of the population, over the next two decades the number of Alzheimer's patients in the US alone will increase from 4-5 million to ten million cases. The social and financial burdens will expand proportionally. Dementia is also seen following loss of blood supply to the brain, for example, following cardiac arrest or some forms of cardiac bypass surgery, also following suffocation or following multiple infarcts in the brain.
Creutzfeld-Jacob disease is a rare disorder, one of a number of transmissible spongiform encephalopathies involving progressive inflammatory neurodegeneration occurring due to the buildup of physicochemically abnormal prion protein. A variant form is seen in cases of variant CJD, a rapidly progressing fatal prion disease mirroring the bovine disorder, “mad cow” disease or bovine spongiform encephalopathy (BSE). The theoretical risk of an epidemic of this disorder in the UK and possibly elsewhere remains, due to the widespread consumption of meat products from BSE-infected cattle during the 1980s and early 1990s.
Traumatic Brain Injury significantly affects over 500,000 individuals annually in the United States alone with a variety of affects from mild concussion to coma and death. 80-85,000 patients annually suffer a head injury that leads to very significant neurological deficit or disability. The estimates of total economic cost place the price tag for TBI at almost $50 billion in the US alone.
Stem cell replacement therapy is seen as a viable treatment option for many diseases including, but not limited to, those described above for which significant cell loss and damage is a cause or consequence. Stem cells can be derived from human tissues at any stage of development, from the early embryo to the adult. Early embryonic stem cells are capable of forming cells from any tissues; however, the cells are likely to form tumours when transplanted. As the tissues develop through the fetal and adult stages, the resident stem cell populations reduce their developmental potential and lose their inherent tumourigenic capacity, becoming somatic stem cells, also known as tissue- or lineage-restricted stem cells. Somatic stem cells are multipotent, that is they are capable of becoming any differentiated cell type from their organ of origin.
Embryonic stem cells can be ‘differentiated’ or selected to form populations of somatic stem cells, which phenotypically resemble somatic stem cells from fetal or adult tissues and therefore lose their tumorigenic potential (for example, WO03-A-000868). Only somatic stem cells (derived from adult or fetal tissues or differentiated from embryonic stem cells) represent, at the current state of knowledge, safe stem cells for cell therapy. The cell lines described in this invention are examples of somatic neural stem cells.
Somatic stem cells from the brain have been proposed as treatments for intractable neurological disorders including Parkinson's diseases, stroke, Huntington's, and and spinal cord injury. Despite early success in anecdotal clinical reports, controlled transplant studies with primary human fetal brain tissue in Parkinson's disease have, however, failed to deliver any consistent benefits [Freed CR, et al., NEW ENGLAND JOURNAL OF MEDICINE 344 (10): 710-719 MAR. 8, 2001; Olanow et al., ANNALS OF NEUROLOGY 54 (3): 403-414 SEPTEMBER 2003] and widespread clinical application of such transplants is in any case constrained by practical and ethical problems.
The heterogeneity of primary human fetal tissues and cells in terms of both cell purity and quality makes the interpretation of such clinical trials difficult. Alternative products can be built on new knowledge of the potential of stem cells as a source of scalable purified cells and tissues for transplantation. For example, following the demonstration that neuroepithelial stem cells and, significantly, clonal cell lines derived from neroepithelial stem cell populations, can restore functional deficits in animal models of neurological diseases [Sinden et al., NEUROSCIENCE 81 (3): 599-608 DECEMBER 1997; WO-A-9710329; Gray et al., PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES 354 (1388): 1407-1421 AUG. 29, 1999], many groups have shown that fetal human neural cells can be expanded for several months in defined media with additional growth factors either as genetically immortalized (by means of the transduction of different immortalizing genes) or as expanded stem cells using specialised culture methods (sometimes referred to as ‘epigenetic’ methods) [Flax et al., NATURE BIOTECHNOLOGY 16 (11): 1033-1039 NOVEMBER 1998; Vescovi et al., EXPERIMENTAL NEUROLOGY 156 (1): 71-83 MARCH 1999]. Neural stem cells, as described in the prior art, have been transplanted into experimental animals and have shown evidence of survival. However, these cells and cell lines are not suitable for use in human patients due to their uncontrolled provenance and manufacture and have not as yet shown evidence of functional efficacy in validated animal models of human neurological disease.
Real clinical and industrial progress in human stem cell transplantation is dependent upon the availability of cell lines with controlled sources and manufacturing that are able to expand quickly and serve as a sustainable resource, available on demand to a broad population of patients. Cell lines could be specific to individual disorders or general across a class of disorders. In order to achieve this goal, cell lines must be generated with appropriate biological characteristics (tissue or cell specific phenotype) and must be sufficiently robust to survive a scaleable manufacturing process to make master and working cell banks of frozen vials of cells from which reproducible, GMP—compliant, clinical lots and commercially viable product batches can be derived. One approach to this problem is to use an immortalizing gene to safeguard the regenerative potential of a cell line and prevent it from entering early senescence. Examples of immortalising genes that have been used to generate neural stem cell lines include: (1) telomerase reverse transcriptase (hTERT) [Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord Roy N S, Nakano T, Keyoung H M, Windrem M, Rashbaum W K, Alonso M L, Kang J, Peng W G, Carpenter M K, Lin J, Nedergaard M, Goldman S A NATURE BIOTECHNOLOGY 22 (3): 297-305 MARCH 2004], (2) SV40 T antigen [Sinden et al., op cit, WO9710329], (3) combinations of SV40 T and hTERT (WO0121790) and myc proteins [U.S. Pat. No. 5,580,777; Flax et al., op cif]. Genetic overexpression of c-myc, a naturally occurring protooncogene that is normally expressed during cell development, is demonstrated in this invention as a means of stably enhancing cell proliferation and preventing changes in cell karyotype, thereby avoiding transformation of the cell phenotype.
Following from considerable experience in developing stem cell lines as therapeutics, we believe the following represent the key features of a human stem cell line for application in the clinic:                Multipotent cells that can develop into specific cells typical of the tissues that are targeted for transplantation.        Cells that are derived from a single founder cell (clonal cells)        Genetically stable cells with normal chromosomes        Cells that have the ability to differentiate into appropriate cell types, in vitro and in vivo.        Cells that can be grown in large numbers and stored        Cells that are safe, particularly not showing tumourigenic potential        Cells whose migration, once implanted, is limited to areas of tissue damage        Cells that are efficacious in recognised animal models        Cells whose provenance is fully documented        
While expansion of stem cells on surface or suspension culture is possible without genetic immortalisation (‘epigentic’ culture), expansion of human neural stem cells is slow and the resulting cultures contain a high proportion of differentiated progeny, which will not survive subculture manipulations [WO99-A-11758, Vescovi et al., op cit]. Further, long term culture of ‘epigenetic’ stem cells may induce chromosomal alterations that may prevent the clinical application of the cell lines. There are no reports of genetic stability of human epigenetic somatic cell lines, although recent reports have indicated chromosomal aberrations in embryonic stem cell lines at greater passage numbers [Draper et al., NATURE BIOTECHNOLOGY 22 (1): 53-54 JANUARY 2004; Inzunza et al., MOLECULAR HUMAN REPRODUCTION 10 (6): 461-466 JUN. 1 2004].
Therefore, the scalability of epigenetically expanded cell lines is limited from an industrial point of view and the product may vary from cell passage to cell passage. The use of an immortalising gene that will not generate a transformed cell phenotype or otherwise influence the stem cell's biological potency or safety but otherwise enhance the cell's industrial scalability and stabilise the karyotype is highly desirable.
Over the past 10 years increasing information has been emerging about the role of myc oncogenes in normal and cell proliferation, differentiation and apoptosis. To maintain cell proliferation Myc and Max proteins dimerise and translocate to the cell nucleus where they serve as transcription factors. Recently Coller et al., [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, 2000; 97:3260-3265] identified 27 genes that were induced, and 9 genes that were repressed by activation of c-myc (cellular myc) in primary human blastocysts. Induced targets included cell cycle genes (e.g. G1 cyclin D2) required to maintain division, and pro-apoptotic genes (e.g. TRAP1) involved in cell death, while repressed targets included genes encoding extra cellular matrix and cytoskeletal proteins, indicating a role for myc in cell adhesion and structure. A recent report has shown that a v-myc (viral myc)-immortalised murine neural stem cell line is able to promote the regeneration of damaged neurons in a Parkinson's disease animal model and promote functional recovery [Ourednik et al., NATURE BIOTECHNOLOGY 20 (11): 1103-1110 NOVEMBER 2002].
The ability of c-myc to maintain cell proliferation makes it a prime candidate for stem cell immortalisation, provided that cell division can be regulated in vivo. For therapeutic use of myc-immortalised cells, a preferred embodiment would permit control over the function of the Myc protein, such that the immortalising protein was not functional after transplanting the cells. This would reduce the risk of overgrowth or tumour formation by the transplanted cells. A preferred conditional form of Myc is a fusion between Myc and the hormone-binding domain of a modified estrogen receptor [Littlewood, et al., NUCLEIC ACIDS RESEARCH, 1995; 23, 1686-1690].